Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1994-04-05
2003-01-07
Priebe, Scott D. (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C435S320100, C514S04400A
Reexamination Certificate
active
06503498
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to methods and compositions for replacement gene therapy, and more particularly relates to adenovirus vectors adapted for delivering functional apolipoprotein A-I (apoA-I) genes to liver cells. It is proposed that the methods and compositions disclosed herein will be applicable to elevating the HDL (high density lipoprotein) form of cholesterol and thus suitable for use in treating atherosclerosis and reducing cardiovascular risk.
2. Description of the Related Art
Epidemiologic data demonstrate an inverse relationship between circulating levels of high density lipoprotein cholesterol (HDL cholesterol) and the incidence of clinically significant atherosclerosis (Miller, 1987; Manninen et al., 1988; Kottke et al., 1986; Gordon et al., 1989). This relationship holds for even small increments of HDL cholesterol, such that each 1 mg/dl increase in HDL cholesterol level is associated with a 2-3% decrement in cardiovascular risk (Gordon et al., 1989). Experimental evidence also supports a protective effect of HDL against atherosclerosis. Cholesterol-fed rabbits treated by infusion of purified homologous HDL are protected against the development of fatty plaques despite unchanged circulating HDL cholesterol levels (Badimon et al., 1989; Badimon et al., 1990; Badimon et al., 1992). This association between HDL cholesterol and the incidence of atherosclerotic vascular disease suggests that strategies to increase circulating HDL could have important clinical application. A modest increase in HDL cholesterol has been observed in patients treated with gemfibrozil (Badimon et al., 1989), an intervention associated with a reduced incidence of cardiac events. Trials intended to specifically assess the effects of intervention to increase HDL cholesterol on the development and progression of atherosclerosis are in progress (Goldbourt et al., 1993; Rubins et al., 1993).
HDL appears to exert its antiatherogenic effect by mediating reverse cholesterol transport, in which cholesterol is mobilized from peripheral tissues and transported to the liver (Eisenberg, 1984; Reichl et al., 1986; Miller, 1990). The small, high density, pre-beta subspecies of HDL, comprised predominantly of apolipoprotein A-1 and phospholipid is thought to act as the physiologic acceptor for cholesterol in the extracellular matrix of peripheral tissues (Reichl et al., 1986). Peripheral availability of this “scavenger” particle appears to be regulated by the rates of synthesis, secretion and catabolism of HDL (Eisenberg, 1984; Reichl et al., 1986; Miller, 1990).
Both clinical and experimental data suggest that the principal protein constituent of HDL, apolipoprotein A-1, mediates the antiatherogenic activity of HDL (Miller, 1987), and that the rate of production of apoA-I is a critical determinant of circulating HDL cholesterol. Families with both heritably deficient (Karathanasis et al., 1983; Vergani et al., 1981; Third et al., 1984; Ordovas et al., 1986) and enhanced (Glueck et al., 1976) apolipoprotein A-1 levels have been identified, and show corresponding alterations in HDL cholesterol. Persons with familial hyperalphalipoproteinemia appear protected from atherosclerosis, while those deficient in apolipoprotein A-1 show accelerated cardiovascular disease. Mice transgenic for a copy of the human apolipoprotein A-1 gene demonstrate accumulation of human apoA-1 in serum, increased circulating HDL cholesterol, and resistance to the atherogenic effects of a high cholesterol diet (Rubin et al., 1991; Walsh et al., 1989; Sorci-Thomas et al., 1988; Rubin et al., 1991). Thus, while the mechanisms regulating the rate of apolipoprotein A-1 synthesis are not clearly defined, genetic factors appear to exert an important effect (Widom et al., 1991).
A potential approach to increasing levels of apolipoprotein A-1 is somatic cell gene therapy. Recently, adenovirus-mediated gene transfer has been investigated as a means of mediating gene transfer into eukaryotic cells and into whole animals (van Doren et al., 1984a; van Doren et al., 1984b; Ghosh-Choudhury and Graham, 1987; Stratford-Perricaudet et at., 1990; Rosenfeld et al., 1991; Rosenfeld et al., 1992). Stratford-Perricaudet et al. (1990) have shown that adenovirus-mediated gene transfer can be used to treat a rare recessive genetic disorder, ornithine transcarbamylase (OTC) deficiency, in newborn mice. Unfortunately, the expression of the ornithine transcarbamylase enzyme in the virus injected mice was comparable to that in normal mice in only 4 out of 17 instances. In one out of 17 instances the level was about half the normal level, and in the remaining 12 out of 17, it was less than 20% of normal. Therefore, the defect was only partially corrected in most of the mice and led to no phenotypic or physiologic change in those mice.
Attempts to use adenovirus to transfer the gene for cystic fibrosis transmembrane conductance regulator (CFTR) into the pulmonary epithelium of cotton rats have also been successful, although it has not been possible to assess the biological activity of the transferred gene in the epithelium of the animals (Rosenfeld et al., 1992). Again, these studies demonstrated gene transfer and expression of the CFTR protein in lung airway cells but showed no physiologic effect. In the 1991 Science article, Rosenfeld et al. showed lung expression of &agr;1-antitrypsin protein but again showed no physiologic effect. In fact, they estimated that the levels of expression that they observed were only about 2% of the level required for protection of the lung in humans, i.e., far below that necessary for a physiologic effect. These results therefore do not demonstrate that adenovirus is able to transfer genes into cells and direct the expression of sufficient protein to achieve a physiologically relevant effect, and would not suggest a usefulness of the adenovirus system for use in connection with apo A-1 gene therapy.
Similarly, the gene for human &agr;
1
-antitrypsin has been introduced into the liver of normal rats by intraportal injection, where it was expressed and resulted in the secretion of the introduced human protein into the plasma of these rats (Jaffe et al., 1992). However, the levels that were obtained were not high enough to be of therapeutic value.
In an alternate approach, a plasmid construct which encodes the human ApoA1 gene has been encapsulated in liposomes and introduced into the liver of rats by direct injection (Frolkis et al., 1991). This method resulted in increased HDL levels in the animals. However, the procedure is invasive, requiring anesthesia and an incision in the abdominal wall in order to introduce the liposome suspension directly into the liver.
Thus, there is clearly a significant need for novel therapeutic approaches that would be applicable to the treatment of diseases involving atherosclerosis. There is a particular need for the development of approaches that can lead to significant increases in HDLc. There is also a particular need for treatment methodologies that do not require surgical intervention, such as direct injection into the liver or modification of hepatocytes ex vivo.
SUMMARY OF THE INVENTION
The present invention addresses one or more of these or other shortcomings in the prior art through the provision of an adenovirus mediated technique for introducing human apoA-1 coding sequences into eukaryotic cells and expression and secretion in liver cells without the need for surgical intervention. The technique of the present invention circumvents many of the problems of the currently available techniques, and is based upon the discovery by the inventors that adenovirus vectors can selectively deliver apoA-1 coding sequences to liver cells and effect expression therein, and thereby achieve a physiologically significant effect.
In view of these observations, somatic cell gene transfer to augment apolipoprotein A-1 expression offers a new and potentially effective therapeutic approach. In an embodiment of the present invention, normal
Gerard Robert D.
Meidell Robert S.
Willard John E.
Board of Regents , The University of Texas System
Fulbright & Jaworski
Priebe Scott D.
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